Histopathological studies of cerebral ischemia, as well as biochemical investigation of ischemia in other tissues, suggest that deterioration of mitochondrial function plays a key role in the development of neuronal death. The proposed project aims to identify important mitochondrial changes using analogous insults in systems of increasing complexity. Initially, isolated mitochondria deprived of oxygen and/or metabolic substrate will be examined for functional changes in respiratory measurements with several substrates, adenine nucleotide content, membrane potential, Ca++ sequestration and the activity of the key enzyme, pyruvate dehydrogenase. Factors believed to influence the deterioration process including ionic composition, pH and lactate content, temperature and certain drugs known to alter metabolic activity will be assessed. The extent to which the changes are reversible will be determined using incubations in which oxygen and/or substrate are restored and/or where ATP is added to the media. Synaptosomes will then be exposed to similar metabolic restrictions, as these represent a model neuronal subfraction which is amenable to manipulation, and for which many of the measurements of isolated mitochondria are possible but in which a cytoplasmic/mitochondrial relationship is maintained. To determine the importance of changes observed in isolated mitochondria, critical parameters will be examined in mitochondria prepared after in vivo insults including ischemia following decapitation and severe hypoxia. Respiratory function and related measurements will also be determined in synaptosomes and brain slices prepared after in vivo insults to provide an assessment of the extent to which mitochondrial alterations are important for maintenance of function in the intact tissue. These studies should provide valuable information on the role of mitochondrial changes in the development and progression of ischemic cell damage, and may provide markers to assess mitochondrial involvement in other human neurological conditions exhibiting selective neuronal loss but where there is little or no information available on pathogenesis.